Overall Component Project Summary/Abstract Hemophilia A is the most common severe congenital bleeding disorder. It is caused by mutations in the F8 gene on the X chromosome that lead to deficiency or complete absence of blood coagulation factor VIII (fVIII). Current management of patients with hemophilia is intravenous infusion of either plasma-derived or recombinant fVIII to prevent bleeding or to treat bleeding after it has occurred. The most significant complication in the management of patients with hemophilia A is the development of inhibitory antibodies (inhibitors) to fVIII. The future standard- of-care of hemophilia A will be gene therapy. In preclinical gene therapy studies, the development of inhibitors has prevented long-term correction of the genetic lesion. We propose to create an Emory Factor VIII Center consisting of a group of highly interactive investigators with diverse research skills and an established interest in the immunogenicity of fVIII. The central hypothesis is that structural determinants in fVIII structure, including its glycans, are responsible for its highly immunogenic behavior. The three projects in this proposal address these themes with aims to determine the relationship of fVIII structure, fVIII glycobiology and fVIII in gene therapy settings to its immunogenicity. In Project 1, ?The Structural Basis for Immune Recognition of Factor VIII?, we seek to identify immunogenic structures that can be modified to produce a less immunogenic fVIII molecule. This will be accomplished by using X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, single-particle negative-stain electron microscopy, surface plasmon resonance spectroscopy and analytical ultracentrifugation to determine the structure of fVIII and fVIII immune complexes. In Project 2, ?The Immunobiology of Factor VIII?, the role of fVIII glycans and the impact of microbiota on anti-?Gal antibody development on inhibitor formation will be determined. Additionally, the immunogenicity of fVIII immune complexes characterized in Project 1 will be evaluated. In Project 3, ?Novel Factor VIII Technologies for the Prevention or Treatment of Factor VIII Inhibitors?, the molecular composition and immune reactivity of bioengineered recombinant infusion products as well as liver-directed AAV and HSC- directed LV gene therapy derived fVIII will be analyzed. Critical design parameters associated with immunogenicity and inhibitor eradiation potential of liver-directed AAV-fVIII gene therapy will be identified. Additionally, the immunogenicity and inhibitor eradicating potential of non-genotoxic HSC-directed LV gene therapy protocols will be evaluated. This program is highly significant because it addresses causal mechanisms for fVIII inhibition and implications for modifying these mechanisms to improve therapy for hemophilia A patients.